Functional antibody responses targeting the Spike protein of SARS-CoV-2 Omicron XBB.1.5 in elderly nursing home residents following Wuhan-Hu-1-based mRNA booster vaccination

The immune effector mechanisms involved in protecting against severe COVID-19 infection in elderly nursing home residents following vaccination or natural infection are not well understood. Here, we measured SARS-CoV-2 Spike (S)-directed functional antibody responses, including neutralizing antibodies (NtAb) and antibody Fc-mediated NK cell activity (degranulation and IFNγ production), against the Wuhan-Hu-1, BA.4/5 (for NtAb), and Omicron XBB.1.5 variants in elderly nursing home residents (n = 39; median age, 91 years) before and following a third (pre- and post-3D) and a fourth (pre- and post-4D) mRNA COVID-19 vaccine dose. Both 3D and 4D boosted NtAb levels against both (sub)variants. Likewise, 3D and 4D increased the ability of sera to trigger both LAMP1- and IFNγ-producing NK cells, in particular against XBB.1.5. In contrast to NtAb titres, the frequencies of LAMP1- and IFNγ-producing NK cells activated by antibodies binding to Wuhan-Hu-1 and Omicron XBB.1.5 S were comparable at all testing times. Stronger functional antibody responses were observed in vaccine-experienced participants compared to vaccine-naïve at some testing times. These findings can contribute to identifying a reliable correlate of protection in elderly nursing home residents against severe COVID-19 and inform future vaccine strategies in this population group.

COVID-19-related morbidity and mortality in elderly nursing home residents have dramatically decreased since the deployment of COVID-19 vaccines.This trend has continued following the emergence of Omicron subvariants following booster vaccination with the primary Wuhan-Hu-1-based vaccine or with (sub)variantadapted vaccine platforms [16][17][18] .The immune effector mechanisms involved in protecting against severe COVID-19 infection in this population group remain poorly defined.In this sense, elderly nursing home residents with frailty and comorbidities frequently develop less robust anti-S antibody and NtAb responses following vaccination compared with seemingly healthy and younger counterparts, which also tend to wane faster [19][20][21][22][23] .In the current study, we measured S-directed functional antibody responses, including NtAb and Fc-mediated NK cell activity, against the ancestral SARS-CoV-2, Omicron BA.4/5 and Omicron XBB.1.5variants in elderly nursing home residents following a third and a fourth mRNA COVID-19 vaccine dose.

Study population
A total of 39 elderly nursing home residents (31 females and 8 males; median age, 91 years; range, 66-103) were enrolled between August 2021 and October 2022.The Charlson comorbidity index (CCI) 24 , originally developed to predict one-year mortality was used to categorize the participants.It contains 19 issues including diabetes, congestive heart failure, peripheral vascular disease, chronic pulmonary disease, mild and severe liver disease, hemiplegia, renal disease, leukaemia, lymphoma, metastatic tumor, and acquired immunodeficiency syndrome (AIDS), each of which is weighted according to their potential influence on mortality.Severe CCI scores indexes are those ≥ 5. Participants in the current study had CCIs of 0-5 (n = 14), 6-10 (n = 23) and > 10 (n = 2).All participants had been previously evaluated for SARS-CoV-2 adaptive immunity following complete regular vaccination with Wuhan-Hu-1-based mRNA vaccines (22 with Comirnaty®, Pfizer/BioNTech and 17 with Spikevax®, Moderna) [25][26][27][28][29] .Whole blood was planned to be collected before and after one (pre-3D and post-3D, respectively) and two (pre-4D and post-4D, respectively) booster vaccine doses for the immunological analyses detailed below.The actual number of specimens from each participant that were tested by the different immunological assays described below is shown in Supplementary table 1.Some participants could not be sampled at a given time due to logistic reasons.Some participants from whom whole blood was collected could not be tested by a given assay due to insufficient volume.The first booster vaccine dose was homologous in all participants, whereas the second was heterologous in 17 participants (third dose with Spikevax® and fourth dose with Comirnaty®).Booster vaccine doses administered were non-adapted, Wuhan-Hu-1 based.The current study was carried out under the epidemiological surveillance competencies of the Valencia Government Health Department (Law 16/2003/May 28 on Cohesion and Quality of the National Health System, and Law 10/2014/ December 29 on Public Health of the Valencian Community), and was approved by the institutional ethical review board of the Foundation for the Promotion of Health and Biomedical Research in the Valencian Community (FISABIO) (Reference, 20210225/02/01).The requirement for informed consent was waived by the ethical review board of FISABIO.Likewise, according to local law and regulations, the publication of the data is exempt from the approval of a research ethics committee.Personal data from nursing homes and residents were deidentified and processed in accordance with European data protection regulations.All methods were performed in accordance with Declaration of Helsinki and the Belmont Report guidelines and regulations.

Statistical analyses
Frequency comparisons for categorical variables were carried out using Fisher's exact test.Quantitative data are reported as medians and interquartile ranges (IQRs).Fold changes in antibody levels between testing times were calculated using non-log-transformed values.Differences between medians were compared using the Mann-Whitney U test for unpaired samples and the Wilcoxon test for paired specimens.The Spearman test was used for correlation analyses.Two-sided exact P values are reported; a P value < 0.05 was considered statistically significant.The analyses were performed using SPSS version 20.0 (SPSS) and STATA 17.0 (StataCorp).

SARS-CoV-2 vaccination and infection status of participants
The timing of booster COVID-19 vaccination and sampling across participants were as follows: pre-3D blood was collected a median of 181 days (range, 178-230) after completion of regular vaccination (two doses); post-3D blood was drawn a median of 17 days (range, 15-17)

Antibody-dependent NK cell-mediated responses against SARS-CoV-2 Wuhan-Hu-1 and Omicron XBB.1.5 spike variants after vaccine boosting
We next measured Fc-dependent S-directed antibody function using an antibody-dependent CD16A-mediated NK cell activity assay.PBMCs obtained from two healthy donors (donor 1 and donor 2) were used in all experiments, yielding similar results (not shown).Results obtained by using PBMCs from donor 1 are detailed below.All participants had detectable NK cell responses at all time points.Overall, the frequencies of LAMP1-and IFNγ-producing NK cells (Fig. 5A,B, respectively) did not differ significantly (P ≥ 0.5) depending on stimulation with Wuhan-Hu-1 or Omicron XBB.1.5S at any testing times; nevertheless, at post-3D, a trend towards higher NK cell frequencies against XBB.1.5compared to Wuhan-Hu-1was observed for both LAMP1 NK cells (72.5% vs. 60.2%;P = 0.05) and IFNγ NK cells (49.3% vs. 43.1%;P = 0.06) (Fig. 5A,B, respectively).Significantly higher frequencies of both LAMP1-or IFNγ-producing NK cells were observed post-3D and post-4D for both (sub)variants compared with pre-3D and pre-4D levels, respectively (Fig. 6, panels A-D).The increase in the frequency of LAMP1-and IFNγ-producing NK cells following the 3D and 4D boosters was more marked for XBB.1.5than for    www.nature.com/scientificreports/Wuhan-Hu-1.When the frequency of functional NK cells activated by antibodies against Wuhan-Hu and Omicron XBB.1.5 was compared between Vac-ex and Vac-n participants, we found no significant differences at any sampling time for either LAMP1 (Fig. 7, panels A and B) or IFNγ-producing NK cells (Fig. 7, panels C and D).

Discussion
The underlying immune effector mechanisms providing protection against severe COVID-19 caused by the ancestral and emerging SARS-CoV-2 (sub)variants in elderly nursing home residents remain to be fully elucidated for both regular and booster vaccination.Here, we aimed to assess the overall impact of 3D and 4D booster doses on Wuhan-Hu-1 and Omicron XBB.1.5S-reactive functional antibody levels, including NtAb and antibodies mediating NK cell activity through Fc.In addition, we sought to determine how these responses compared across SARS-CoV-2-experienced and naïve participants.Importantly, XBB.1.5has been displaced by newly emerged Omicron subvariants such as BA.2.86; however, NtAb levels in sera from vaccinated individuals have been shown to display similar or even slightly higher efficiency against XBB.1.5compared with current dominating variants, including BA.2.86, irrespective of whether a breakthrough infection with the latter (sub) variant had or had not occur 33,34 .Several major observations were made in the current study.First, both 3D and 4D boosted, to some extent, NtAb levels against both Wuhan-Hu-1 (and also anti-RBD total antibodies) and Omicron XBB.1.5.This effect was notably more marked following 3D as compared with 4D.Nevertheless, median www.nature.com/scientificreports/NtAb titres post-4D were significantly higher than post-3D for all (sub)variants.Likewise, the increase in the frequency of both LAMP1-and IFNγ-producing NK cells following the 3D booster was more pronounced than after the 4D booster, particularly against XBB.1.5.Since antibody levels wane over time, the simplest explanation accounting for these findings is the longer time elapsed between vaccination and collection for the 4D as compared to the 3D; nevertheless, we cannot rule out that pre-existing SARS-CoV-2 specific antibodies limits humoral immunity boosting 35,36 .In keeping with the aforementioned observation, the anti-RBD IgG AI, which directly correlates with the level of antibody functionality 37 , significantly increased after the 3D but remained unchanged following the 4D.Second, while median NtAb titers against Wuhan-Hu-1 were significantly higher than those against Omicron XBB.1.5 at all testing times, the frequencies of LAMP1-and IFNγ-producing NK cells activated by plasma antibodies binding either to Wuhan-Hu-1 or Omicron XBB.1.5S were comparable.Moreover, the correlation between NtAb titres and LAMP1-and IFNγ-producing NK cell frequencies against Omicron XBB.1.5 was moderate and weak against Wuhan-Hu-1.These findings reinforce the idea that antibodies mediating FcR activation either bind to more conserved S epitopes across SARS-CoV-2 (sub)variants than NtAb do or require a less stringent binding affinity for S epitopes than NtAb to be functionally effective 5,[9][10][11][12][13][14][15] .Regardless of the underlying mechanistic explanation, antibodies mediating FcR NK cell activation seem to be less sensitive to SARS-CoV-2 escape by newly emerging variants than NtAb.This may have important implications regarding the COVID-19 vaccine and therapeutic moAb design.Third, a trend towards higher NtAb levels in Vac-ex compared with Vac-n after 3D, regardless of the SARS-CoV-2 (sub)variant, was observed pre-3D but not at other time points.A similar finding was previously reported by our group in apparently healthy individuals following regular COVID vaccination for NtAb binding the Wuhan-Hu-1 S and the Omicron BA.1 S protein 14 .In turn, the frequencies of both LAMP1-and IFNγ-producing NK cells activated by plasma antibodies were rather similar for Vac-Ex and Vac-n at all testing times.These data challenge the assumption that hybrid immunity provides stronger functional antibody responses than vaccination alone [38][39][40][41][42] , at least in elderly nursing home residents receiving one or two booster vaccine doses.Fourth, our study provides further evidence of the increased ability of Omicron XBB.1.5 to escape from NtAb compared with preceding Omicron lineages, such as BA.4/5 33 .The current study is not without limitations.First, its relatively limited sample size, in particular the small number of Vac-n after 3D, limits the robustness of some of the analyses performed.Second, as previously described 14 , a surrogate antibody-mediated NK cell activation assay was used.Third, antibody-mediated NK cell and NtAb responses against currently dominant Omicron subvariants (i.e., BA.2.86) were not evaluated.While these subvariants do not appear to outperform XBB.1.5 in terms of evasion from NtAb, despite incorporating additional mutations in the RBD domain 33,34 , their ability to escape antibodies mediating NK cell activity is unknown.Fourth, the SARS-CoV-2 (subvariant) infecting the participants was inferred, based upon the timing of infection, but not proven by genome sequencing approaches.This precluded meaningful analyses addressing the potential impact of the infecting (sub)variant on the strength of the functional antibody responses measured.Moreover, we could not ascertain whether participants were infected one or more times by different lineages.Furthermore, categorization as Vac-ex or Vac-n on the basis of the presence or absence of detectable anti-SARS-CoV-2 N antibodies, respectively, may be erroneous due to waning of such antibody specificities.Fifth, due to logistic constraints, FC-mediated NK cell responses against Omicron BA.4/5 were not assessed.Sixth, there was no control group.Finally, we wish to highlight a major limitation of the study; that is the is the different time elapsed between receipt of 3D and 4D and post-3D and post-4D immunological testing.This precluded to properly compare the impact of both booster vaccine doses on functional antibody titers.Unfortunately, logistic issues precluded testing within an identical timeframe.In summary, our data revealed that that both 3D and 4D boostered functional antibody responses and further stressed the fact that NK cell Fc-dependent functional activity against Omicron XBB.1.5 is relatively conserved in fully vaccinated participants boosted with Wuhan-Hu-1-based mRNA vaccines.The data presented herein can contribute to identifying a reliable correlate of protection in elderly nursing home residents, potentially helping to recognize individuals at particular risk of severe COVID-19 and inform future vaccine strategies in this population group.
after receipt of 3D (median time between pre-3D and post-3D, 49 days; range, 20-73); pre-4D blood was obtained a median of 331 days (range, 198-339) post-3D and post-4D blood was collected a median of 112 days (range, 87-116) after 4D (median time elapsed between pre-4D and post-4D, 122 days; range, 116-127).SARS-CoV-2-experienced (Vac-ex) participants were those who had acquired SARS-CoV-2 infection; whereas SARS-CoV-2 naïve (Vac-n) were those who seemingly had not contracted SARS-CoV-2 infection.Categorization as Vac-ex or Vac-n was based upon the detection of anti-SARS-CoV-2-N IgG in plasma, a record of a positive RT-PCR result in nasopharyngeal specimens or both at the different time points of the study.At the time of study initiation (pre-3D), there were 27 Vac-n and 12 Vac-ex while only eight participants remained SARS-CoV-2-naïve at the end of the study period (post-4D).According to the dominant SARS-CoV-2 (sub)variant at a given time, 10 Vac-ex had been presumably infected by pre-Omicron (sub)variants, whereas the remaining 21 Vac-ex had been infected by Omicron (sub)variants (pre-XBB.1).

Figure 1 .
Figure 1.Anti-Receptor binding domain (RBD) total antibodies (in log 10 in BAU/mL) in plasma from elderly nursing home residents.A antibody levels prior to and after one (pre-3D and post-3D, respectively) or two (pre-4D and post-4D, respectively) COVID-19 vaccine booster doses.B Antibody levels in vaccinated/SARS-CoV-2-experienced (Vac-ex) and vaccinated/SARS-CoV-2-naïve subjects (Vac-n) at the different sampling times.The number of plasma samples available for analyses at the different sampling times is shown.Bars indicate medians and IQRs.P values for differences are shown.

Figure 2 .
Figure 2. Anti-Receptor binding domain (RBD) IgG avidity in plasma from elderly nursing home residents.Total antibodies.A Anti-RBD IgG avidity indexes (%) before and after one (pre-3D and post-3D, respectively) or two (pre-4D and post-4D, respectively) COVID-19 vaccine booster doses.B Anti-RBD IgG avidity indexes (%) in vaccinated/SARS-CoV-2-experienced (Vac-ex) and vaccinated/SARS-CoV-2 naïve (Vac-n) at the different sampling times.The number of plasma samples available for analyses at the different sampling times is shown.Bars indicate medians and IQRs.P values for differences are shown.

Figure 3 .
Figure 3. Neutralizing antibody titres (reciprocal IC 50 ) against the spike protein of Wuhan-Hu-1 (A), Omicron BA.4/5 (B), and Omicron XBB.1.5(C) in elderly nursing home residents before and after one (pre-3D and post-3D, respectively) or two (pre-4D and post-4D, respectively) COVID-19 vaccine booster doses.The number of plasma samples analysed at the different sampling times is shown.Bars indicate medians and IQRs.P values for differences are shown.

Figure 4 .
Figure 4. Neutralizing antibody titres (reciprocal IC 50 ) against the spike protein of Wuhan-Hu-1, Omicron BA.4/5 , and Omicron XBB.1.5 in elderly nursing home residents before and after one (pre-3D and post-3D, respectively) or two (pre-4D and post-4D, respectively) COVID-19 vaccine booster doses, according to their SARS-CoV-2 infection status (experienced/Vac-ex or naïve/Vac-n).The number of plasma samples analysed at the different sampling times is shown.Bars indicate medians and IQRs.P values for differences are shown.

Figure 5 .
Figure 5.Comparison of antibody-dependent NK cell-mediated responses against SARS-CoV-2 Wuhan-Hu-1 and Omicron XBB.1.5spike variants in elderly nursing home residents before and after one (pre-3D and post-3D, respectively) or two (pre-4D and post-4D, respectively) COVID-19 vaccine booster doses.Frequencies of LAMP1-(lysosomal-associated membrane protein 1) and IFN (interferon) γ-producing NK cells are shown in panels A and B, respectively.The number of participants analysed at the different sampling times is shown.Bars indicate medians and IQRs.P values for differences are shown.

Figure 6 .
Figure 6.Impact of COVID-19 booster vaccination on antibody-dependent NK cell-mediated responses against SARS-CoV-2 Wuhan-Hu-1 and Omicron XBB.1.5spike variants in elderly nursing home residents.The frequencies of LAMP1 (lysosomal-associated membrane protein 1)-producing NK cells against Wuhan-Hu-1 and Omicron XBB.1.5 are shown in (A) and (B), respectively.The frequencies of IFN (interferon) γ-producing NK cells against Wuhan-Hu-1 and Omicron XBB.1.5 are shown in (C) and (D), respectively.The number of participants analysed at the different sampling times is shown.Bars indicate medians and IQRs.P values for differences are shown.

Figure 7 .
Figure 7. Antibody-dependent NK cell-mediated responses against SARS-CoV-2 Wuhan-Hu-1 and Omicron XBB.1.5spike variants in elderly nursing home residents according to their SARS-CoV-2 infection status (experienced/Vac-ex or naïve/Vac-n).The frequencies of LAMP1-(lysosomal-associated membrane protein 1)-producing NK cells against Wuhan-Hu-1 and Omicron XBB.1.5 at the different testing times are shown in (A) and (B), respectively.The frequencies of IFN (interferon)γ-producing NK cells against Wuhan-Hu-1 and Omicron XBB.1.5 are shown in (C) and (D), respectively.The number of participants analysed at the different sampling times is shown.Bars indicate medians and IQRs.P values for differences are shown.